qPCR

qPCR= QUANTITATIVE POLYMERASE CHAIN REACTION (=real time PCR)

Real-Time Vs Traditional PCR:


Real-Time chemistries allow for the detection of PCR amplification during the early phases of the reaction. Measuring the kinetics of the reaction in the early phases of PCR provides a distinct advantage over traditional PCR detection. Traditional methods use agarose gels for detection of PCR amplification at the final phase or end-point of the PCR reaction. Real-Time PCR makes quantitation of DNA and RNA easier and more precise than past methods.

·       

There are 3 basic principles of qPCR:
1)SYBR Green
2)TaqMan Probe
3) Molecular Bacon

All these metods aresimilar. They quantitatively determine expression of protein-at the end of experiment we know how many copies of  some gene was done or concentartion RNA in our sample.  qPCR use double-stranded DNA-binding dyes as reporters.

 

SYBR Green

A DNA-binding dye binds to all double-stranded (ds) DNA in PCR, causing fluorescence of the dye. An increase in DNA product during PCR therefore leads to an increase in fluorescence intensity and is measured at each cycle, thus allowing DNA concentrations to be quantified. However, dsDNA dyes such as SYBR Green will bind to all dsDNA PCR products, including nonspecific PCR products (such as Primer dimer). The SYBR Green is excited using blue light (λ_max = 488 nm) and it emits green light (λ_max = 522 nm).

1. The reaction is prepared as usual, with the addition of fluorescent dsDNA dye.
2. The reaction is run in a quantitative PCR instrument, and after each cycle, the levels of fluorescence are measured with a detector; the dye only fluoresces when bound to the dsDNA (i.e., the PCR product). With reference to a standard dilution, the dsDNA concentration in the PCR can be determined.

TaqMan Probe

The method relies on a DNA-based probe with a fluorescent reporter at one end and a quencher of fluorescence at the opposite end of the probe. The close proximity of the reporter to the quencher prevents detection of its fluorescence; breakdown of the probe by the 5' to 3' exonuclease activity of the Taq polymerase breaks the reporter-quencher proximity and thus allows unquenched emission of fluorescence, which can be detected after excitation with a laser. An increase in the product targeted by the reporter probe at each PCR cycle therefore causes a proportional increase in fluorescence due to the breakdown of the probe and release of the reporter.

1.  The PCR is prepared as usual (see PCR), and the reporter probe is added.
2. As the reaction commences, during the annealing stage of the PCR both probe and primers anneal to the DNA target.
3. Polymerisation of a new DNA strand is initiated from the primers, and once the polymerase reaches the probe, its 5'-3'-exonuclease degrades the probe, physically separating the fluorescent reporter from the quencher, resulting in an increase in fluorescence.
4. Fluorescence is detected and measured in a real-time PCR machine, and its geometric increase corresponding to exponential increase of the product is used to determine the quantification cycle (C_q) in each reaction.

Fluorescent probes can be used in multiplex assays—for detection of several genes in the same reaction—based on specific probes with different-coloured labels, provided that all targeted genes are amplified with similar efficiency. 
 

 Molecular Bacon

in this method we have hairpin probe. On 3' end is quencher, on 5' fluorescent reporter. whean they are near, they don`t shine. During PCR reaction this Molecular Bacon bound on the DNA, the quencher and fluorescent reporter are divorced, that result in an increase in fluorescence.

For better understanding i recomend this video:

http://www.youtube.com/watch?v=kvQWKcMdyS4

Real-Time PCR Applications:

• Viral Quantitation
• Quantitation of Gene Expression
•  Array Verification
• Drug Therapy Efficacy
•  DNA Damage measurement
•  Quality Control and Assay Validation
• Pathogen detection
•  Genotyping